About the project: microRNAs (miRNAs) are 22 nucleotide small non-coding RNAs that regulate translation, deadenylation and decay of their target mRNAs. miRNAs have recently taken central stage in biology due to their fundamental roles in animal development, human disease and cancer. Typically, two RNase III family members, Drosha and Dicer, process the stem-loop structure of miRNA precursors sequentially. My recent work has uncovered a novel processing pathway where miRNA maturation skips the Dicer step and instead enters into an alternative Ago2-dependent pathway. This finding challenges a long-held assumption that Dicer is essential for miRNA maturation. However, Ago2-mediated cleavage is just the initial step of this novel pathway and little is known about the molecular machinery and the mechanisms underlying the final steps of miRNA maturation. My preliminary results suggest that after Ago2-mediated cleavage, miR-451 is uridylated and processed to its mature form by exonucleolytic trimming, a mechanism that has recently been reported as a widespread method to refine mature miRNA length. Whether the trimming and uridylation machinery is common to both the canonical and Ago2-dependent miRNA processing pathways is a question that I will explore in this project. To uncover the miRNA trimming (Aim 1) and uridylation machinery (Aim 2), during the mentored phase of the award I will focus on the analysis of miR-451 processing because processing of this Ago2-dependent miRNA requires pervasive uridylation and extensive trimming. Moreover, miR-451 is conserved across vertebrates and plays an essential role both in erythrocyte maturation and in metabolic regulation of glioblastoma tumors making the results of this project relevant for human therapeutics. Later, in the independent period of the award I will capitalize on the discovery of the uridylation and trimming machinery to analyze their role not only in Ago2-dependent miRNA production but also in canonical small RNA biogenesis and turnover during vertebrate development (Aim 3). The use of zebrafish embryos will be crucial to perform these analyses in a genome-wide manner. The use of the zebrafish model organism removes the restrictions of using single tissue or cell types, which have limited repertoire of small RNAs. To accomplish all of these objectives I will combine biochemistry, mass spectrometry, genetics and high- throughput sequencing. The experiments in this proposal will identify an evolutionarily conserved machinery to process small regulatory RNAs in vertebrates. The results derived from this project will be instrumental to understand how pervasive modifications of the 3'-end by tailing and trimming affects miRNA-target selection in vivo during early embryogenesis. miRNAs play a key role during early embryogenesis clearing maternal mRNAs and mechanisms controlling miRNA turnover will have a direct impact in embryonic development. Furthermore, given the relevance of trimming in miR-451 processing, the discovery of the underlying machinery will potentially set the framework for therapeutic intervention in human hematopoietic disorders and cancer. About the candidate: Dr. Cifuentes will continue his training as Postdoctoral associate in the Genetics Department at Yale University, where he will enjoy both state-of-the-art facilities and the interaction with his mentors and other scientific leaders of th field. In this environment, Dr. Cifuentes will use the Career Development Award to fulfill his short-term goal of acquiring specific training in the areas of i) computational biology and ii) mass- spectrometry. This will allow him to obtain high quality publications and a strong presence in the microRNA field independent of his previous mentors. These accomplishments will establish the framework to achieve the long-term goal of understanding how alternative processing pathways modulate miRNA function and the impact of these pathways in development and human disease. Furthermore, Dr. Cifuentes plans to expand his previous teaching and mentoring experience by participating in structured courses and workshops. This will allow him to learn innovative and effective ways to teach biology and progress to become a more rounded scientist and mentor. Dr. Cifuentes is fully committed to obtain an appointment as a tenure track junior faculty in academia. It is fully expected that Dr. Cifuentes will be competitive for such group leader appointments at the completion of his Career Development Award, tailored to enhance his scientific and mentoring skills.